A signal is amplified by making a cars beam from an observed body and a reference beam which is a portion of a super continuum beam and has a frequency of ωAS=2ωP−ωST interfere with each other and taking out the signal from an interference beam of the cars beam and the reference beam.
|
1. An optical apparatus comprising:
a first beam having a frequency ωP;
a unit for generating a super continuum beam;
a split unit for dividing the super continuum beam into a second beam and a third beam with the frequency ωP as a boundary;
the second beam having a frequency lower than the frequency ωP maintaining a relationship of being coherent with the first beam;
the third beam which is a reference beam having a frequency of ωAS=2ωP-ωST, the frequency being higher than the frequency ωP and which is a beam that does not irradiate an observed sample;
an optical system configured to make the first beam and the second beam coincide with each other substantially coaxially;
a mechanism configured to adjust phases of the first beam or the second beam;
an object lens configured to focus the first beam and the second beam;
an object lens configured to detect a cars beam generated from an observed sample;
an interference optical system configured to make the cars beam and the third beam interfere with each other coaxially;
a photo-detector configured to detect an interference beam; and
an arithmetic unit configured to process a signal from the optical detector.
2. The optical apparatus according to
3. The optical apparatus according to
4. The optical apparatus according to
an image display unit configured to display an image based on the arithmetic unit.
5. The optical apparatus according to
6. The optical apparatus according to
7. The optical apparatus according to
|
The present application claims priority from Japanese patent application JP 2012-034635 filed on Feb. 21, 2012, the content of which is hereby incorporated by reference into this application.
This application is related to U.S. application Ser. No. 13/546,055 filed on Jul. 11, 2012, the disclosure of which is hereby incorporated by reference.
The present invention relates to an optical apparatus which requires an optical resolving power, particularly relates to an optical apparatus which focuses a light beam and acquires a response signal by relatively changing a position of irradiating an observed body with the light beam.
A Raman spectroscopic microscope is very effective for observing a sample related to an organism. In the Raman spectroscopic microscope, an observed object is irradiated with a laser beam focused thereto, and a generated Raman scattering beam is detected. The Raman scattering beam is shifted from a wavelength of an excitation beam in a frequency thereof, and a spectrum is obtained by a spectroscope or the like. Positions of the observed object and an irradiation beam are changed relative to each other and the observed object is scanned by which spectroscopic spectra at respective positions can be obtained. An image can be formed based on the spectra. Raman spectra at respective observation positions reflect a vibrational excitement state of a molecule which is present at the positions, and are characteristic to the molecule. When a cell of an organism is observed by making use of the characteristic of the spectra, a distribution of organism molecules in a tissue is known.
The Raman scattering described above has a weak intensity of the scattering beam obtained, and therefore, time is taken for measurement. As a system of obtaining a strong scattering beam, there is a spectrometry which uses nonlinear Raman scattering referred to as CARS (Coherent Anti-Stokes Raman Scattering). Raman scattering can be obtained even by the method, and a vibrational state of a molecule can be known. A pulse laser having a high peak power is used for generating CARS. Thereby, a signal which is remarkably stronger than an a signal of Raman scattering is obtained, that is, a signal of a high signal to noise ratio is obtained, and a measurement time period can remarkably be shortened.
CARS is light emission by a third-order polarization, and a pumping beam, a stokes beam, and a probe beam are needed for generating CARS. Generally, a pumping beam substitutes for a probe beam in order to reduce a number of light sources. In this case, an induced third-order polarization is expressed as follows.
Equation 1
PAS(3)(ωAS)=|χr(3)(ωAS)+χnr(3)|EP2(ωP)E*S(ωS) (1)
Here, notation χr(3)(ωAS) designates a resonance term of a molecular vibration of an electric third-order susceptibility, and notation χnr(3) designates a non-resonance term thereof. Also, electric fields of the pumping beam and the probe beam are expressed as EP, and an electric field of the stokes beam is expressed as ES. The non-resonance term does not have frequency dependency. An asterisk attached to the shoulder of ES of Equation 1 indicates complex conjugate. An intensity of a CARS beam is expressed as follows.
Equation 2
ICARS(ωAS)∝|PAS(3)(ωAS)|2 (2)
An explanation will be given of a mechanism of generating the CARS beam in reference to an energy level diagram of a molecule (
Researches of spectra with regard to various kinds of molecules have been carried out since the Raman scattering was discovered in 1928, and accumulation of data is progressed. Therefore, it is preferable to identify a molecule in reference to the spectra data. CARS beam is expressed by Equations 1 and 2, and a portion in proportion to a Raman scattering spectrum is Im[χr(3)(ωAS)]. This is a complex part of the resonance term, which interferes with the non-resonance term χnr(3) as described above, and therefore, the Raman scattering spectrum cannot directly be obtained by the spectrum obtained by CARS.
A development of a method of extracting the Raman scattering spectrum from the CARS spectrum is an important problem, and various systems have been developed therefor (J. P. R. Day, K. F. Domke, G. Rago, H. Kano, H. Hamaguchi, E. M. Vartiainen, and M. Bonn “Quantitative Coherent Anti-stokes Scattering (CARS) Microscopy,” J. Phys. Chem. B, Vol. 115, 7713-7725 (2011)). For example, according to a maximum entropy method which is a method of recovering a phase spectrum from an intensity spectrum, a complex part of a resonance term is calculated by carrying out a mathematical calculation. Or, there is also a method making use of interference (C. L. Evans, E. O. Potma, X. S. and Xie, “Coherent Anti-Stokes Raman Scattering Spectral Interferometry: Determination of the Real and Imaginary Components of Nonlinear Susceptibility χ(3) for Vibrational Microscopy,” Opt. Lett. Vol. 29, 2923-2925 (2004)). According to the method, a CARS signal is generated by simultaneously focusing a pumping beam and a stokes beam to an observed sample by a condenser lens. On the other hand, a non-resonant CARS signal is obtained by irradiating a separate sample which generates the non-resonant CARS signal with a pumping beam and a stokes beam. The non-resonant CARS signal is made to be a local beam and the both CARS signals are interfered with each other. The local beam of the non-resonance signal is made to be a circularly polarized beam by a λ/4 plate, and a polarizing direction of the CARS beam from the observed sample is rotated by 45 degrees by a λ/2 plate. An interference beam of the beams is separated into two different linearly polarized beams, and the respective beams are subjected to spectrometry by a spectroscope. When an electric field of CARS from the observed sample is designated by notation EAS (ω) and an electric field of the local beam is designated by notation ELO, respective interference signals are expressed as follows.
Equation 3
SC(ω)=|ELO|2+EAS(ω)|2+2|ELOEAS(ω)|cos Φ(ω) (3)
Equation 4
SS(ω)=|ELO|2+|EAS(ω)|2+2|ELOEAS(ω)|sin Φ(ω) (4)
Here, notation Φ(ω) expresses a phase difference between the local beam and the CARS signal beam, and is expressed as Φ(ω)=ωτ+θS(ω)+θinst(ω). Notation COT designates an optical path difference between the two beams, notation θS(ω) designates a phase difference by a resonant beam, and notation θinst(ω) designates a phase difference originated from an apparatus. |ELO|2 and |EAS(ω)|2 in Equations 3 and 4 can be calculated by cutting off one of them. Therefore, tan Φ(ω) can be calculated and also Φ(ω) can be determined from Equations 3 and 4. First, ωτ+θinst(ω) is determined by measuring a sample which generates only the non-resonant CARS beam as an observed sample. Next, an observed sample which generates the resonant CARS is measured. θS(ω) can be determined thereby, and therefore, the complex part of the resonance component can be calculated as |EAS(ω)|sin θS(ω). Thereby, what corresponds to the Raman scattering spectrum can be obtained.
The above-described method is a method of determining ωτ+θinst(ω) accurately by using the sample which generates the non-resonant CARS beam. However, when there are known a peak frequency of the CARS spectrum which is obtained previously and a frequency by which the spectrum becomes flat in a case of small frequency dependency of θinst(ω), there may be set θinst(ω) as an initial value for realizing the spectrum. In this case, a reference sample which generates the non-resonance CARS beam is not needed.
There is a Raman scattering spectrum region (1800 through 800 cm−1) which is referred to as a fingerprint region as a spectrum region which is sensitive to a molecular structure. It is preferable to obtain a spectrum of a similar region also in detecting a CARS beam. According to the system introduced in J. P. R. Day, K. F. Domke, G. Rago, H. Kano, H. Hamaguchi, E. M. Vartiainen, and M. Bonn “Quantitative Coherent Anti-stokes Scattering (CARS) Microscopy,” J. Phys. Chem. B, Vol. 115, 7713-7725 (2011), a spectrum width of the stokes beam for excitement is about 140 cm−1, and the system cannot cover the region. There is introduced a system of using a photonic fiber as a light source for compensating for the drawback in M. Okuno, H. Kano, P. Leproux, V. Couderc, J. P. R. Day, M. Bonn, and H. Hamaguchi, “Quantitative CARS Molecular Fingerprinting of Single Living Cells with the Use of the Maximum Entropy Method,” Angew. Chem. Int. Ed. Vol. 49.6773-6777 (2010). There is generated a broadband beam which is referred to as Super Continuum Beam (SC beam) by irradiating a photonic fiber with an extremely short pulse laser.
There is “noninvasiveness” which is regarded as useful when a living cell is observed by CARS. Extremely short pulse beams of a pumping beam and a stokes beam are simultaneously irradiated in order to generate CARS. Generally, a wavelength which is not absorbed by a living cell is used in wavelengths of the both excited beams used in CARS. Therefore, it can be said that the “noninvasiveness” is established in a state of a low peak power, and the cell is not damaged. However, when the peak power is increased excessively, a multiphoton process is brought about, and there is a possibility of effecting an influence on the cell. It is preferable that the peak power of irradiating the living cell is low even when the wavelength is “noninvasiveness”. It is a problem of the present invention to improve a signal to noise ratio of a weak CARS signal which is generated under a condition of restraining the peak power of the excited beam and realize a high spatial resolving power.
An optical apparatus for resolving the problem described above is realized by an optical apparatus including a first laser beam having a frequency ωP, a second laser beam having a frequency ωST maintaining a relationship of being coherent with the first laser beam, a third laser beam which is a portion of a super continuum beam and has a frequency of ωAS=2ωP−ωST as a reference beam, an optical system of making the first laser and the second laser beam coincide with each other substantially coaxially, a mechanism of adjusting phases of the first beam and the second beam, an object lens of focusing the first laser beam and the second laser beam, an object lens of detecting a CARS beam generated from an observed sample, an interference optical system of making the CARS beam and the third laser beam interfere with each other, a photo-detector of detecting an interference beam, and an arithmetic unit of processing a signal from the photo-detector. In a case where a display unit is integrated to the optical apparatus, the optical apparatus further includes the display unit of displaying an image based on information of the arithmetic unit.
The wavelength of the first or the second laser beam may be made to be variable in order to acquire a CARS spectrum by the optical apparatus.
A band of the second laser may be made to be a broadband and a spectroscope may be used for detecting a spectrum of the interference beam in order to acquire the CARS spectrum by the optical apparatus.
An optical filter may be installed in an optical path of the reference beam in order to improve a spatial resolving power of the optical apparatus.
According to the present invention, a signal can be amplified by making the CARS beam from the observed body and the reference beam interfere with each other and taking out the signal from the interference beam even in a state of reducing a power density of the irradiated laser beam. Thereby, the noninvasiveness can be maintained, the signal to noise ratio can be improved, and the spatial resolving power can be improved.
An explanation will be given of the best mode for embodying an optical apparatus according to the present invention in reference to the drawings as follows.
Also a laser beam of a high frequency region reflected by the dichroic mirror 257 is an SC beam, and is used as a reference beam (third beam) including the frequency (ωAS=2ωP−ωST). The reference beam is collimated by lenses 258 and 206, and transmits through an optical filter 220. A laser beam which is partially shielded or decreased by the optical filter 220 transmits through a polarized light beam splitter 216 and a Fresnel rhomb wavelength plate 217 having an effect of a λ/4 plate and is returned to the Fresnel rhomb wavelength plate 217 by a mirror 218. The mirror 218 is used for adjusting the optical path length. A laser beam which transmits through the Fresnel rhomb wavelength plate 217 becomes a p-polarized beam, is reflected by the polarized beam splitter 216, and is directed to the beam splitter 213.
Beams having different polarizing directions are incident on the half beam splitter 213 from two directions, each of the beams is split in two directions, and interference beams are emitted in two directions. A method referred to as phase diversity detection is used for detecting |EAS(ω)|. A Fresnel rhomb wavelength plate 221 having an effect of a λ/2 plate an optical axis of which is inclined by 22.5 degrees is installed for an interference beam emitted in a right direction of the half beam splitter 213 on paper face, and the beam is converged onto a spectroscope which is placed at a focal point position of a condenser lens 215. A polarized beam splitter 223 is installed at an optical path in front of the spectroscope, the interference beam is separated into components in an s direction and in a p direction, and the respective beams are detected by a spectroscope 106 and a spectroscope 108. Here, the observed body is made to be a point body which is present on an optical axis of a focal point face, and a complex amplitude of the CARS beam of the observed object, and a complex amplitude of the reference beam are respectively made to be EAS(ω) and ELO. When a differential signal of respective frequencies of the respective beams at the spectroscope 106 and the spectroscope 108 is designated by notation IC(ω), the difference signal is expressed as IC(ω)=α|EAS(ω)|·|ELO|cos Φ(ω). Notation α designates a coefficient including signal amplification and a spectroscope efficiency, and notation Φ(ω) designates a phase difference of the CARS beam from the observed object and the reference beam. A Fresnel rhomb wavelength plate 222 having an effect of a 214 plate an optical axis of which is inclined by 45 degrees is inserted to an interference beam which is emitted in an upper direction of the half beam splitter 213 on paper face. The interference beam converged by a condenser lens 214 is detected by spectroscopes 105 and 107. The interference beam is separated into an s-polarized beam and a p-polarized beam by a polarized beam splitter 224 which is installed at a midway, and thereafter, detected by the respective spectroscopes. Here, when a differential signal of respective frequencies of the spectroscopes 105 and 107 is designated by notation IS(ω), the differential signal is expressed as in IS(ω)=α|EAS(ω)∥ELO|sin Φ(ω). Only interference components are detected in Ic(ω) and IS(ω). An arithmetic unit 109 carries out a calculation shown below.
Equation 5
I(ω)=√(IC2(ω)+IS2(ω))=α|EAS(ω)∥ELO| (5)
I(ω) is configured by a form proportional to an amplitude of the CARS beam of the observed object and an amplitude of the reference beam. Therefore, I(ω) in a form of amplifying |EAS(ω)| can be obtained by enlarging |ELO| which has no wavelength dependency. Generally, a spectrum of an SC beam is not flat, and therefore, it is necessary to carry out a correction which uses the amplitude spectrum of the SC beam in order to acquire further accurate spectrum I(ω). Next, a complex component [I(ω) sin θS(ω)] of a resonance term is obtained by calculating a phase difference which is generated by a resonant beam, that is, by the method of using an interference described above, or by extracting a complex component of a resonance term of the CARS beam by the maximum entropy method. A detector of CCD or the like may be used for detecting by a spectroscope. Numeral 110 designates a display unit, and there is carried out a display with a display position corresponding to a scan position of the observed body 202. A distribution of a molecule can be known by displaying I(ω) at a frequency position that is characteristic to a vibration of the molecule.
Assume that a point body as the observed body on a focal point face that is present at a location remote from an optical axis by a distance a on x-axis is irradiated with a pumping beam and a stokes beam, and a CARS beam is generated. Two-dimensional amplitude point image distribution functions of the pumping beam and the stokes beam are respectively designated by notations hP and hS, and a two-dimensional amplitude point image distribution function of the generated CARS beam on a spectroscope is designated by notation hAS. There is formed a point image distribution hAS(x-Ma, y) centering on a position remote from an optical axis by Ma on the respective four spectroscopes by designating a magnification of an optical system by notation M. Also, simultaneously, the reference beam is configured by a point image distribution hAS(x, y) centering on an optical axis. When a two-dimensional point image distribution function at a certain wavelength of the CARS beam which is formed from an output of Equation 5 as a result of interference is designated by notation hCARS, a two-dimensional amplitude point image distribution function |hCARS (a, 0)| which adopts an absolute value on x-axis is expressed as follows.
Equation 6
|hCARS(a,0)|=|hP(a,0)|2|hS(a,0)∥∫∫hAS(x−Ma,y)hAS(x,y)dxdy| (6)
A surface integral is carried out on the detector. Since a coherent optical system is configured, optically, the point image distribution function is configured by squaring Equation 6. However, also Im[χr(3)(ωAS)] proportional to a concentration of a substance is configured by a squared form, and therefore, a square sign is not attached to Equation 6. A point image distribution function in an ordinary optical system which does not use a CARS beam and does not use an interference optical system becomes |hP (a, 0)|2, in, for example, a wavelength of a pumping beam. In comparison with the ordinary point image distribution function, in Equation 6, the point image distribution function of the CARS beam is narrowed since a part of an excitation beam is multiplied by |hS (a, 0)| and also multiplied by an integration term in Equation 6, and then a resolving power is improved. In a case of arranging an optical filter at the reference beam, a two-dimensional amplitude point image distribution function hAS(x, y) of the reference beam in an integration of Equation 6 can be narrowed. Therefore, the point image distribution function of the CARS beam is further narrowed, and the spatial resolving power is improved.
Although the embodiment of
In the embodiment described above, there is adopted a system of interfering linearly polarized beams orthogonal to each other, and the phase diversity detection is carried out. As other system, there is also a method of making the CARS beam from the observed body and the reference beam orthogonal to each other in a state of circularly polarized beams and interfering the CARS beam and the reference beam. For example, the CARS beam is made to be a right-handed circularly polarized beam and the reference beam is made to be a left-handed circularly polarized beam. When the optical axis of a polarizer for detecting the interfered beam is set to 0, 45, 90, 135 degrees, there are acquired beams having phase differences of 0, 90, 180, 270 degrees relatively. A signal expressed by Equation 5 can be obtained by combining the signals, and an effect similar to that of the embodiment described above can be achieved.
In an embodiment of
A spectroscope is not used in an embodiment of
Equation 7
S(ωAS)=|ELO|2+|EAS(ωAS)|2+2|ELOEP2(ωP)ES(ωS)|{[χnr(3)+Reχr(3)(ωAS)] cos δ+[Imχr(3)(ωAS)] sin δ} (7)
Notation ELO designates an amplitude of a non-resonant CARS beam from the reference beam generating sample, and notation EAS(ωAS) designates an amplitude of a CARS beam generated from the observed sample. When the phase difference δ is modulated by the phase modulator 227, the phase difference δ is converted into an intensity modulation as shown in Equation 7. A value proportional to Im χr(3) (ωAS) can be acquired by configuring the detecting signal by taking out a sine component by phase sensitive detection by the lock-in amplifier 113. When a wavelength of the pumping beam is changed, also ωAS is changed, and a spectrum can be acquired. A distribution of a specific molecule can also be measured by making the wavelength of the pumping beam coincident with a resonance wavelength of the molecule and scanning the observed sample 202 by the scanning mechanism 102.
Incidentally, although in the present embodiment, the wavelength of the first beam is made to be variable, the wavelength of the stokes beam which is the second beam may be made to be variable. A spectrum can be acquired without using a spectroscope by making the wavelength of the first or the second beam variable.
Although in any of the embodiments described above, the example of scanning the observed body is shown, the scanning is not limited to the observed body, but the optical system may be scanned.
According to the present invention, there can be acquired an image using a CARS beam and having a high resolving power, and the present invention can be applied to an optical apparatus for noninvasively measuring a distribution of a molecule of an organism or a change in the distribution.
Watanabe, Koichi, Shirai, Masataka, Kimura, Shigeharu
Patent | Priority | Assignee | Title |
10216064, | Jul 20 2016 | Deutsches Elektronen-Synchrotron Desy | Optical parametric waveform synthesizer and method for synthesizing optical waveforms |
Patent | Priority | Assignee | Title |
4405237, | Feb 04 1981 | The United States of America as represented by the Secretary of the Navy | Coherent anti-Stokes Raman device |
6934020, | Jul 03 2001 | EVIDENT CORPORATION | Laser microscope |
7388668, | Jun 09 2004 | President and Fellows of Harvard College | Phase sensitive heterodyne coherent anti-Stokes Raman scattering micro-spectroscopy and microscopy |
7688505, | Dec 09 2005 | Auburn University | Simultaneous observation of darkfield images and fluorescence using filter and diaphragm |
8120772, | Nov 03 2008 | The United States of America as represented by the Secretary of Commerce, The National Institute of Standards and Technology | Method for NRB-free nonlinear vibrational spectroscopy and miscroscopy |
8482738, | May 04 2007 | APE ANGEWANDTE PHYSIK AND ELEKTRONIK GMBH | Method and optical arrangement for generating a nonlinear optical signal on a material which is excited by an excitation field, and use of the method and of the optical arrangement |
20080059135, | |||
20100309465, | |||
20110128538, | |||
20120050733, | |||
20120092665, | |||
JP2006276667, |
Executed on | Assignor | Assignee | Conveyance | Frame | Reel | Doc |
Nov 07 2012 | KIMURA, SHIGEHARU | Hitachi, LTD | ASSIGNMENT OF ASSIGNORS INTEREST SEE DOCUMENT FOR DETAILS | 029574 | /0279 | |
Nov 08 2012 | WATANABE, KOICHI | Hitachi, LTD | ASSIGNMENT OF ASSIGNORS INTEREST SEE DOCUMENT FOR DETAILS | 029574 | /0279 | |
Nov 21 2012 | SHIRAI, MASATAKA | Hitachi, LTD | ASSIGNMENT OF ASSIGNORS INTEREST SEE DOCUMENT FOR DETAILS | 029574 | /0279 | |
Jan 07 2013 | Hitachi, Ltd. | (assignment on the face of the patent) | / |
Date | Maintenance Fee Events |
May 16 2016 | ASPN: Payor Number Assigned. |
Oct 11 2018 | M1551: Payment of Maintenance Fee, 4th Year, Large Entity. |
Dec 19 2022 | REM: Maintenance Fee Reminder Mailed. |
Jun 05 2023 | EXP: Patent Expired for Failure to Pay Maintenance Fees. |
Date | Maintenance Schedule |
Apr 28 2018 | 4 years fee payment window open |
Oct 28 2018 | 6 months grace period start (w surcharge) |
Apr 28 2019 | patent expiry (for year 4) |
Apr 28 2021 | 2 years to revive unintentionally abandoned end. (for year 4) |
Apr 28 2022 | 8 years fee payment window open |
Oct 28 2022 | 6 months grace period start (w surcharge) |
Apr 28 2023 | patent expiry (for year 8) |
Apr 28 2025 | 2 years to revive unintentionally abandoned end. (for year 8) |
Apr 28 2026 | 12 years fee payment window open |
Oct 28 2026 | 6 months grace period start (w surcharge) |
Apr 28 2027 | patent expiry (for year 12) |
Apr 28 2029 | 2 years to revive unintentionally abandoned end. (for year 12) |